title: Localization of the anti-cancer peptide EGFR-lytic hybrid peptide in human pancreatic cancer BxPC-3 cells by immunocytochemistry.
abstract: Cationic lytic-type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti-cancer epithelial growth factor receptor (EGFR)-lytic hybrid peptide, a 32-amino-acid peptide composed of an EGFR-binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR-lytic hybrid peptide in BxPC-3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR-lytic peptide compared with non-treated cells. In confocal laser scanning microscopy, immunostaining of EGFR-lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR-lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR-lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR-lytic peptide; however, no effect on cell death after EGFR-lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR-lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death.
authors.affiliation: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Japan Yoshidakonoecho, Sakyoku, Kyoto city, Kyoto 606-8501, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Ohara
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kohno
authors.forename: Koji
authors.initials: K
authors.lastname: Kawakami
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D017209:Apoptosis; D061945:Caspase Inhibitors / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D008933:Mitochondrial Swelling; D010190:Pancreatic Neoplasms; D021381:Protein Transport; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; C586251:EGFR-lytic peptide; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004317:Doxorubicin; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: anti-cancer peptide; hybrid peptide; immunocytochemistry; lytic type peptide